Betulinic acid is a pentacyclic triterpene. It has several botanical sources, but can also be chemically derived from betulin, a substance found in abundance in the outer bark of white birch trees (Betula alba). Betulinic acid has been found to selectively kill human melanoma cells (Nature Medicine, Vol.1 (10), 1995, WO 96/29068). The cytotoxic potential of betulinic acid was tested using three human melanoma cell lines, Mel-1, -2, and 4. The growth of all of the cell lines was inhibited significantly by treatment with betulinic acid. The effectiveness of betulinic acid against melanoma cancer cells was also tested using athymic mice. It seems to work by inducing apoptosis in cancer cells.
The anti-cancer activity of betulinic acid and some of its derivatives has also been demonstrated using mouse sarcoma 180 cells implanted s.c. in nude mice (JP 87,301,580), inhibition of growth of P388 lymphocytic leukemia cells in vitro (Choi.Y-H et al., Planta Medica Vol.XLVII,511-513,1988) and inhibiting growth of cancer cells, particularly by inhibiting ornithine decarboxylase (Yasukawa, K et al, Oncology 48:72-76,1991; WO 95/04526).
Recently, we reported the anti-leukemia and anti-lymphoma activity of betulinic acid and its derivatives with ED.sub.50 values in the range of approximately 0.5 to 4.0 .mu.g/ml. and also reported anti-prostate, anti-lung and anti-ovarian cancer activity of betulinic acid and betulinic acid derivatives with ED.sub.50 values in the range of approximately 0.6 to 6.8 .mu.g/ml, to 1.3 to 7.7 .mu.g/ml, and 0.4 to 8.1 .mu.g/ml respectively (see U.S. application Ser. No. 09/040,856 filed on Mar. 18, 1998 now U.S. Pat. No. 6,048,847, the subject matter of which is incorporated by reference).
The lupanes, and specifically betulinic acid, have been reported to be effective anti-inflammatory agents. (Sotomatsu s., et al; Skin and Urology 21:138,1959 and Inoue H. et al; Chem.Pharm.Bull 2:897-901, 1986). The anti-inflammatory activity of betulinic acid is, at least in part, due to its capacity to inhibit enzymes involved in leukotriene biosynthesis, including 5-lipoxygenase. Betulinic acid and its derivatives have been tested for their ability to inhibit 5-lipoxygenase activity by monitoring their ability to inhibit conversion of linoleic acid to 5-HETE. On pre-incubating with the enzyme, betulinic acid inhibits the production of 5-HETE by approximately 40%. (WO 95/04526).
Angiogenesis is the growth of new microvessels, a process that depends mainly on locomotion, proliferation, and tube formation by capillary endothelial cells. During angiogenesis, endothelial cells emerge from their quiescent state and can proliferate as rapidly as bone marrow cells, but unlike the bone marrow, angiogenesis is usually focal and of brief duration. Pathologic angiogenesis, while still a focal process, persists for months or years. The angiogenesis that occurs in diseases of ocular neovascularisation, arthritis, skin diseases, and tumors rarely terminates spontaneously and has until recently, been difficult to suppress therapeutically. Therefore, the fundamental goal of all antiangiogenic therapy is to return foci of proliferating microvessels to their normal resting state, and to prevent their regrowth (Cancer: Principles & Practice of Oncology, Fifili Edition, edited by Vincent T. DeVita, Jr., Samuel Hellman, Steven A. Rosenberg. Lippincott-Raven Publishers, Philadelphia.COPYRGT. 1997).
Although the molecular mechanisms responsible for transition of a cell to angiogenic phenotype are not known, the sequence of events leading to the formation of new vessels has been well documented (Hanahan, D., Science 277, 48-50, 1997). The vascular growth entails either endothelial sprouting (Risau, W., Nature 386, 671-674, 1997) or intussusception (Patan, S., et al; Microvasc. Res. 51, 260-272, 1996). In the first pathway, the following sequence of events may occur: (a) dissolution of the basement of the vessel, usually a postcapillary venule, and the interstitial matrix; (b) migration of endothelial cells toward the stimulus; (c) proliferation of endothelial cells trailing behind the leading endothelial cell (s); (d) formation of lumen (canalization) in the endothelial array/sprout; (e) formation of branches and loops by confluencelanastomoses of sprouts to permit blood flow; (f) investment of the vessel with pericytes; and (g) formation of basement membrane around the immature vessel. New vessels can also be formed via the second pathway: insertion of interstitial tissue columns into the lumen of preexisting vessels. The subsequent growth of these columns and their stabilization result in partitioning of the vessel lumen and remodelling of the local vascular network.
The rationale for antiangiogenic therapy is that progressive tumor growth is angiogenesis-dependent (Folkman, J.; N.Engl.J.Med., 285, 1182, 1971). The switch to the angiogeneic phenotype appears to be an independent event that occurs during the multistage progression to neoplasia. The angiogenic switch itself, while relatively sudden and well localized, is nonetheless a complex process. This phenotype is currently understood in terms of a shift in the net balance of stimulators and inhibitors of angiogenesis, during which inhibitors are downregulated.
Endothelial cell survival and growth are driven by tumor derived mitogens and motogens. These findings have led to a model of tumor growth in which the endothelial cell compartment and the tumor cell compartment interact with each other. They not only stimulate each other's growth, but if the endothelial cells are made unresponsive to angiogenic stimuli from the tumor cells, by administration of a specific endothelial inhibitor, both primary tumors and metastatic tumors can be held dormant at a microscopic size (O'Reilly M S, et al; Cell, 19, 315, 1994 and O'Reilly, MS et al; Nature Med, 2, 689, 1996). One could take advantage of this difference between endothelial cells and tumor cells by administering an angiogenesis inhibitor together with conventional cytotoxic chemotherapy up to the point at which the cytotoxic therapy would normally be discontinued because of toxicity or drug resistance. The angiogenesis inhibitor(s) could then be continued for years, to maintain either stable disease or tumor dormancy. Such combinations of antiangiogenic and cytotoxic therapy in tumor-bearing animals have been curative, whereas either agent alone is merely inhibitory.
Eicosapentanoic acid (EPA), a n-3 polyunsaturated fatty acid found in fish and marine animals is a precursor for eicosanoids. EPA taken by man competes with arachidonic acid for inclusion in cycloxygenase and lipoxygenase pathways. Exposure of endothelial cells to EPA in vitro inhibited their capacity to form tubes--an essential aspect of the angiogenic process and substantially decreases their ability to break through an artificial basement membrane (Matrigel) in vitro (Kanayasu T et al, Lipids 1991;26:271-276). Repeated topical application of EPA in eye drops significantly suppressed the resulting neovascularisation and associated inflammation in induced immunogenic keratitis in rabbit corneas (Verbey N L J, et al, Curr Eye Res 1986; 7:549-557).
A similar effect is observed with gamma linolenic acid (GLA). A number of studies demonstrate that GLA slows cancer growth in animals. GLA also slows angiogenesis. (Ormerod L D et al, Am.J. Pathol. 1990; 137:1243-1252 and Verbey N L J, et al, Curr Eye Res 1986; 7:549-557). The mechanism of this effect is not clear, although GLA supplementation increases certain lipoxygenase products which potently inhibit 5-lipoxygenase enzyme as well as 12-lipoxygenase. (Vanderhoek J Y et al, Biochem. Pharmacol. 1982, 31:3463-3467; Miller C C et al, Prostaglandins 1988; 35: 917-938; Miller C C et al, J. Invest. Dermatol. 1991; 96:98-103; Ziboh V A, Am J Clin Nutr, 1992, 52:39-450). It is conceived that GLA impedes angiogenesis by reducing lipoxygenase activity and thereby slowing the mitotic response of endothelial cells.
The mitotic response of endothelial cells may be mediated, at least in part, by products of the lipoxygenase pathway. When endothelial cells are incubated in culture with fetal calf serum, they multiply at rate that can be quantified. Co-incubation with the lipoxygenase inhibitors NDGA or ETYA substantially reduces their multiplication rate. Although ETYA also inhibits cycloxygenase, the specific cycloxygenase inhibitor indomethacin does not influence endothelial mitosis, suggesting that it is lipoxygenase activity which is crucial to endothelial multiplication. (Setty B N Y et al, Biochem. Biophys. Res. Commn., 1987; 144:345-351).
It has also been shown that some arachidonate derived products of 5-lipoxygenase enhances collagenase production by cancer cells in vitro by inhibiting this enzyme (Reich et al Biochem. Biophys. Res. Commn., 1989; 160:559-564). Collagenase and other lytic enzymes are secreted by endothelial cells to enable migration through basement membranes. Collagenase induction seems to be an obligatory step in angiogenesis, as the metalloproteinase inhibitor 1,10 phenanthroline, which inhibits collagenase but not most other proteolytic enzymes, blocks angiogenesis in vitro (Montesano R, Orci L; Cell 1985; 42:469477).
Certain cancer cells are known to adhere to E-Selectin via E-Selectin ligands on their cell surface and this event is one component of the metastasis process. Betulinic acid and its derivatives interfere with Selectin binding. Betulinic acid inhibited P-Selectin binding to 2,3, sLex, a chemical known to bind to P-Selectin, with an IC.sub.50 of 125 uM. It also inhibited P-Selectin binding to HL-60 cells in a dose-dependent way with an IC.sub.50 of 0.75 mM. Betulinic acid and derivatives also significantly interfere with the binding to colon cancer cells, LS 174T to E-Selectin (WO 95/04526).
Several anti-inflammatory agents including fish oil rich in EPA, DHA and DHLA, gamma linolenic acid (GLA) and cromolyn significantly suppress inflammation, angiogenesis and tumor invasiveness. Angiogenesis can thus be viewed as having pathways common with the inflammatory process. Without being limited to a particular mechanism of action, it is surmised that betulinic acid and its derivatives impede angiogenesis by inhibiting 5-lipoxygenase and this subsequently retards tumor growth.
Several angiogenesis inhibitors like Angiostatin, Endostatin, inhibitors of Fibroblast Growth Factor and inhibitors of Vascular Endothelial Growth Factor were found to be antiangiogenic by the virtue of their endothelial cell inhibitory activity (Cancer: Principles & Practice of Oncology, Fifth Edition, 3075-3085, edited by Vincent T. DeVita et al. Lippincott-Raven Publishers, Philadelphia .COPYRGT. 1997).
The human umbilical vein endothelial cells (HUVECS) have been used extensively to study the biology and pathobiology of the human endothelial cells including their role in angiogenesis (Exp. Cell Res., 225, 171-185, 1996). Due to the functional and structural endothelial cell heterogeneity between and within species, and the time consuming and difficult isolation and culture methods, a model endothelial cell line has been used that provides standardization of observations. The spontaneously transformed and fully characterized human umbilical vein endothelial cell line ECV304 provides a reproducible and biologically relevant experimental model system for in vitro angiogenesis (In Vitro Cell Dev. Biol., 25, 265-274, 1990). ECV304 cells maintain a stable functional phenotype throughout serial cultivations and exhibit consistent response in in vitro angiogenesis assays (Exp. Cell Res., 225, 171-185, 1996). We have used the ECV304 cell line and report for the first time the antiangiogenic activity of betulinic acid and its derivatives.